Herpes Simplex Virus (HSV) infects most human beings and not only causes troublesome genital disease but also can result in necrotizing encephalitis or blinding keratitis in normal hosts and severe, often disseminated infections of neonatal and immunodeficient patients. The viral genes involved in such invasion and dissemination remain largely unknown, but it is clear that HSV-2 has an invasiveness potential superior to HSV-1. We have found the greatest such type specific difference after chorioallantoic membrane (CAM) inoculation of the chicken embryo, where HSV-2 is a million-fold more virulent. We recently discovered that, unlike other HSV-1 strains, HSV-1 ANG behaves like HSV-2 and invades the CAM mesoderm, generalizes and kills embryos. Additionally, we lave localized, cloned and transferred to a normal HSV-1 strain the two virulence regions involved, each independently conferring full CAM virulence. The involved genes map within the ANG EcoR1 A and F regions which we have tentatively named "INV-I" and "INV-II" respectively. The cloned recombinant viruses F1 and F2, which incorporated sequences from the EcoR1 F1 INV-II region, are also a thousand-fold more neuroinvasive and virulent after footpad inoculation of a mammalian host, the mouse. Extensive fine restriction mapping of these virulent recombinants reveals an ANG DNA insert within a 3.7 kb region (0.334-0.360 mu) encoding parts of 2 HSV-1 genes: glycoprotein g Beta and the p40 capsid protein. We now plan to fully characterize the INV I and II virulence functions by extensive subcloning and CAM virulence studies to allow: 1) determination of precise physical location followed by b) determination of the mRNA transcripts, nucleotide sequences and proteins responsible for virulence. Detailed comparative studies of the pathogenesis of CAM and mouse infection with the parental strains and the virulent recombinants will be conducted to provide an understanding of the pathogenic action(s) of the genes identified. We also will determine whether the HSV-2 sequences homologous to these ANG genes are related or identical to those of ANG and, if so, responsible for the enhanced invasiveness. If the involved ANG genes are found to be unrelated to those found in HSV- 2 we will then study whether certain HSV-2 glycoproteins (gPs) contribute to HSV-2 invasiveness of HSV-2. The genes for HSV-2 gPs (gC, gG, g Beta) will be inserted into HSV-1 and monoclonal antibodies used to select chimeric recombinants, expressing the specific HSV-2 gP, which will then be studied for their virulence. In sum, these studies should define at least two HSV genes found to be involved in invasiveness and provide important new insights into the molecular mechanisms of HSV virulence.